In 2015, the Apicomplexan Molecular Physiology Section examined the molecular basis of nutrient and toxin uptake by human erythrocytes infected with the malaria parasite, P. falciparum. We previously used in vitro selections to generate HB3-leuR1, a parasite line resistant to the toxin leupeptin. This protease inhibitor is considered membrane-impermeant, but must enter infected cells to kill the parasite. HB3-LeuR1 exhibited reduced uptake of not only leupeptin but other solutes that enter infected cells via the plasmodial surface anion channel (PSAC), a broad permeability nutrient and ion channel identified by our group. There was a single mutation in the clag3 gene linked to PSAC activity, but the relationship between this nonsynonymous mutation (A1210T), altered PSAC activity, and leupeptin resistance was unclear. In the present studies, we used computational studies to identify a conserved amphipathic domain including the A1210 residue on the CLAG3 protein. Helical wheel analysis revealed strict segregation of polar and hydrophobic residues to opposite faces of the putative alpha-helix, consistent with a transmembrane domain that lines a water-filled pore. We therefore used site-directed mutagenesis and DNA transfection of wild-type malaria parasites to introduce the A1210T mutation without leupeptin selection. The transfected parasite exhibited altered solute selectivity through PSAC, changes in channel pharmacology, and increased tolerance to leupeptin challenge. These findings support a direct contribution of CLAG3 to PSAC activity and organic solute transport. They also reveal the molecular basis of a novel antimalarial resistance mechanism that involves reduced channel-mediated uptake of toxins and drugs. These findings provide insights into PSAC structure-function and should inform antimalarial drug development programs about the risk of acquired resistance. Infection and Immunity 83(6):2566-74. (2015).